The present invention relates to human growth factor receptors.
The formation of blood capillaries occurs in a number of important biological processes, either physiological, such as organ development and wound healing, or pathological, such as tumor growth. While the sequence of events leading to neovascularization has been characterized morphologically, the molecular mechanisms by which this process occurs are still poorly understood. The control of growth in the capillary endothelium appears to be very tightly controlled, since these cells normally form a static monolayer whose proliferation is triggered in the angiogenic process. The normally quiescent nature of the endothelial cells may be explained in part by the apparent lack of endothelial cell growth factors to plasma. The major endothelial cell mitogens in fact are not found in plasma, although they are present in extracts of almost all tissues studied and in many normal and tumor cell lines as well. Therefore, the localized induction of rapid endothelial cell proliferation may involve the release of endothelial cell mitogens from cells in response to environmental cues.
The best characterized of the endothelial cell mitogens are a family of polypeptide growth factors, including basic fibroblast growth factor (bFGF), also known as heparin-binding growth factors for their high affinity to heparin. Basic FGF has been purified from most mesoderm- or neuroectoderm-derived tissues or cells. Structural studies have shown that bFGF is a single chain polypeptide made of 146 amino acids, which can also exist in NH.sub.2 -terminally truncated forms missing the first 10-20 amino acids. The truncated forms of bFGF are as potent as native bFGF, as demonstrated by radioreceptor binding and biological assays. In addition, modifications of the purification protocols by substitution of neutral for acidic extraction from homogeneized tissue and inclusion of protease inhibitors have yielded a longer 154-residue form. The observed microheterogeneity of FGFs seems to be due, at least in part, to partial proteolysis near the amino termini that occurs either in vivo or during purification. However, because the various forms appear to be equally active, the microheterogeneity is probably physiologically irrelevant.
Basic FGF seems to have been extremely well conserved through evolution. For example, bovine and human bFGF differ in only two of their 146 amino acids, giving an overall amino acid sequence homology of 98.7%. Related to bFGF is acidic FGF (aFGF), which shares a 55% total sequence homology with bFGF. Acidic FGF is a 140-amino acid polypeptide that can also exist in a NH.sub.2 -terminally truncated form missing the first 6 amino acids. As expected from their high degree of homology, both basic and acidic FGF seem to interact with the same cell-surface receptors. This explains their common range of target cells and spectrum of biological activity.
Recently, Lee et al (Science, 245, 57-60, 1989) have described the purification, from chicken embryos, of a new membrane protein able to bind specifically basic FGF. On the base of its biochemical characteristics and on homology with other known receptors, this new protein is thought to be a basic FGF receptor. In the same article the authors describe the isolation of a chicken full-length cDNA clone coding for the described protein. The nucleotide sequence of this clone is however not disclosed.
Lee et al noted that the chicken bFGF receptor shows a considerable amino acid similarity to a previously identified human polypeptide sequence which is the product of the flg gene (Ruta et al, Oncogene, 3, 9-15, 1988). Like the chicken bFGF receptor, the flg molecule seems to be a tyrosine kinase, shows a typical hydrophobic transmembrane region and consequently it is likely that the described human sequence could be part of the human bFGF receptor.
The amino acid sequence of the flg molecule was deduced by Ruta et al by the translation of an open reading frame of a partial cDNA clone. The flg cDNA sequence, according to Rutal et al was obtained by low-stringency screening of a human endothelial cell cDNA library using as probe a DNA fragment coding for a tyrosine kinase oncogene. Nevertheless, the flg cDNA clone, described to date, is only partial and lacks the nucleotide sequence coding for the extracellular portion of the human bFGF receptor.
In summary, the only amino acid or nucleotide sequences available prior to the present invention were those corresponding to the complete chicken bFGF receptor and to a portion of the human basic FGF receptor. The complete extracellular amino acid sequence of the human bFGF receptor, which is responsible for the specific binding of human basic and acidic FGFs, was unknown.